Project Summary/Abstract Affecting approximately 2 million people in the US each year, concussion or mild traumatic brain injury (mTBI) has only recently been recognized as a major health issue. This injury is by no means `mild' since it induces persisting cognitive dysfunction in many individuals. Moreover, there is increasing concern that there may be a period of vulnerability after one mTBI, during which time a second mTBI may induce greatly exaggerated pathophysiological effects. Despite the impact of mTBI, the field has only just begun to identify mechanisms, especially with regards to repetitive mTBI. Nonetheless, there is mounting evidence that traumatic axonal injury (TAI) plays an important role in single and repetitive mTBI. The goal of the proposed work is to study TAI mechanisms that are relevant to single and repetitive mTBI. This will be done using an integrated approach of in vitro experimentation with computational modeling. Our well-characterized in vitro TAI model has been used to make seminal observations identifying mechanisms of TAI that have subsequently been found in large animal and human TBI. Using this model it is proposed to examine the fundamental biomechanical thresholds that govern microtubule (MT) failure by examining the non-linear viscoelastic response to stretch injury. Since all the individual failure events cannot be accessed in situ, a quantitative ultrastructural computational model of the axon will be used to guide and interpret experimental work. Aim 1 will focus on identifying acute mechanisms of TAI failure as a function of strain and strain rate. Electron microscopy, immunofluorescence and changes in intra-axonal calcium will be used to examine mechanical breaking of MTs and unbinding of the microtubule stabilizing protein tau. A novel computational model, based on the three-dimensional ultrastructure of the axon, identify injury thresholds at the level of the whole axon and nanometer level taking into account MT-tau binding, tau diffusion and MT pretension. Aim 2 will concentrate on the spatio-temporal evolution of MT depolymerization, phosphorylation / translocation of tau and axon degeneration or recovery following TAI. The same in vitro measures from Aim 1 will be used over a temporal time course using identified injury threshold parameters. Aim 3 will examine axon vulnerability to a second injury. This vulnerability will be assessed in relation to MT stability, tau translocation and an axons ability to recover following the first injury. A strain and strain rate threshold for MT failure will be determined. A computational model following the spatiotemporal evolution of phosphorylated densities of tau, MT polymerization kinetics and stress distributions will be developed to predict vulnerability criterion based on stress, strain rate, MT length and time to a second injury. It is anticipated that the data generated will reveal biomechanical thresholds and new mechanisms of single and repetitive traumatic axonal injury that will be relevant to mild TBI in humans.